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Presence of endogenous ent-kaurene in a microsomal preparation from Cucurbita maxima L. Endosperm and implications for kinetic studies of ent-kaurene oxidase

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Abstract

Microsomal and soluble cell-free extracts prepared from liquid endosperm of Cucurbita maxima L. were found to contain high concentrations of endogenous ent-kaurene and ent-kaurenol by gas chromatography-mass spectrometry-chemical ionization with deuterated internal standards. Increases in the levels of ent-kaurenol, ent-kaurenoic acid, and ent-7α-hydroxykaurenoic acid are correlated with a decline in the amount of endogenous ent-kaurene following a 10 min incubation of microsomes with NADPH and FAD. The rate of oxidation of radiolabeled ent-kaurene by the microsomal fraction was determined, and the need to account for endogenous substrate is shown. Endogenous ent-kaurene present in soluble extracts had the effect of diluting the [14C]ent-kaurene synthesized from [14C]mevalonic acid, resulting in reduced specific radioactivity of the product. The dilution of [14C]ent-kaurene was more pronounced in extracts with higher endogenous ent-kaurene levels or when the reactions were run in the presence of O2 and NADPH. Evidence is presented that suggests differential metabolism of endogenous ent-kaurene and radiolabeled ent-kaurene in both microsomal and soluble extracts.

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Abbreviations

Kaurene:

ent-kaur-16-ene

MVA:

mevalonic acid

kaurenol:

ent-kaur-16-en-19-ol

kaurenoic acid:

ent-kaur-16-en-19-oic acid

EtOAc:

ethyl acetate

MeOH:

methanol

GC-MS-CI:

gas chromatography-mass spectrometry-chemical ionization

13-OH KA:

ent-13-hydroxykaur-16-en-19-oic acid

7α-OH kaurenoic acid:

ent-7α-hydroxykaur-16-en-19-oic acid

kaurenal:

ent-kaur-16-en-19-al

Me(x):

methyl ester of x

TMS(x):

trimethylsilyl ether or ester of x

GA(x):

gibberellin A(x)

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Author notes

  1. Jan P. Hazebroek

    Present address: Pioneer Hi-Bred Int'n., Inc., 7300 N.W. 62nd Avenue, 50131-1004, Johnston, Iowa, USA

Authors and Affiliations

  1. Department of Botany and Plant Pathology, Purdue University, 47907, West Lafayette, Indiana, USA

    Jan P. Hazebroek, Susan E. Schechter & Ronald C. Coolbaugh

  2. USDA, Agricultural Research Service, Biosciences Research Laboratory, State University Station, 58105, Fargo, North Dakota, USA

    Jan P. Hazebroek & James D. Metzger

Authors
  1. Jan P. Hazebroek
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  2. Susan E. Schechter
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  3. James D. Metzger
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  4. Ronald C. Coolbaugh
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Hazebroek, J.P., Schechter, S.E., Metzger, J.D. et al. Presence of endogenous ent-kaurene in a microsomal preparation from Cucurbita maxima L. Endosperm and implications for kinetic studies of ent-kaurene oxidase. J Plant Growth Regul 12, 109–115 (1993). https://doi.org/10.1007/BF00189640

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  • DOI: https://doi.org/10.1007/BF00189640

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